Method of using prophenins-antibiotic peptides

ABSTRACT

Peptide-based compounds containing multiple proline residues are useful as preservatives and in preventing, treating, or ameliorating microbial infection, especially Gram-negative bacterial infection in animals and plants, in treating conditions characterized by the presence of LPS. These compounds are of the formula ##STR1## including the N-terminal acylated and/or C-terminal amidated or esterified forms thereof 
     wherein each of A 1 , A 5 , A 1O , A 14  and A 20  is independently Ala, Gly or Ser; 
     each of A 2 , A 4 , A 7 , A 9 , A 11 , A 12 , A 13 , A 16 , A 17 , A 19  and A 22  is independently a hydrophobic amino acid selected from the group consisting of Ile, Leu, Val, Phe and Met; 
     each of A 3  and A 8  is independently a neutral polar amino acid selected from Asn and Gln or is a hydrophobic amino acid selected from the group consisting of Ile, Leu, Val, Phe and Met; 
     each of A 6 , A 18  and A 21  is independently a basic amino acid selected from Arg, Lys, and Har; and 
     wherein A 15  is Trp or a basic amino acid selected from Lys, Arg and Har; 
     wherein n is an integer of 1-6; and the antimicrobial or LPS-binding fragments thereof. Recombinant materials for the production of these peptides are also disclosed.

This invention was made with funding from NIH Grant No. A122839. TheU.S. Government has certain rights in this invention.

This application is a continuation of application Ser. No. 08/22,798filed 5, Apr. 1994.

TECHNICAL FIELD

The invention relates to the field of antibiotic peptides. Inparticular, the invention concerns peptides, some of which can beisolated from porcine leukocytes, that are particularly effectiveagainst Gram-negative bacteria and can mitigate the symptoms of septicshock.

BACKGROUND ART

One of the defense mechanisms against infection by both animals andplants is the production of peptides that have antimicrobial andantiviral activity. Various classes of these peptides have been isolatedfrom tissues both of plants and animals. One well known class of suchpeptides is the tachyplesins which were first isolated from thehemocytes of the horseshoe crab as described by Nakamura, T. et al. JBiol Chem (1988) 263:16709-16713. This article described the initialtachyplesin isolated from the Japanese species, Tachyplesin I, which isa 17-amino acid amidated peptide containing four cysteine residuesproviding two intramolecular cystine bonds. In a later article by thisgroup, Miyata, T. et al. J. Biochem (1989) 206:663-668, extends thestudies to the American horseshoe crab and isolated a secondtachyplesin, Tachyplesin II, consisting of 17 residues amidated at theC-terminus, also containing four cysteine residues and twointramolecular disulfide bonds. Two additional 18-mers, calledpolyphemusins, highly homologous to Tachyplesin II and containing thesame positions for the four cysteine residues, were also isolated.Polyphemusin I and Polyphemusin II differ from each other only in thereplacement of one arginine residue by a lysine. All of the peptideswere described as having antifungal and antibacterial activity. A laterarticle by Murakami, T. et al. Chemotherapy (1991) 37:327-334, describesthe antiviral activity of the tachyplesins with respect to vesicularstomatus virus; Herpes Simplex Virus I & II, Adenovirus I, Reovirus IIand Poliovirus I were resistant to inactivation by Tachyplesin I.Morimoto, M. et al. Chemotherapy (1991) 37:206-211, found thatTachyplesin I was inhibitory to Human Immunodeficiency Virus. Thisanti-HIV activity was found also to be possessed by a synthetic analogof Polyphemusin II as described by Nakashima, H. et al. AntimicrobialAgents and Chemotherapy (1992) 1249-1255. Antiviral peptides have alsobeen found in rabbit leukocytes as reported by Lehrer, R. I. et al. J.Virol (1985) 54:467-472.

Another class of antimicrobial and antiviral peptides, the "protegrins"have been isolated from porcine leukocytes as reported by the presentapplicants in a paper by Kokryakov, V. N. et al. FEBS (1993)337:231-236. An additional paper disclosing cationic peptides fromporcine leukocytes was published by Mirgorodskaya, O. A. et al. FEBS(1993) 330:339-342. Storici, P. et al. Biochem Biophys Res Comm (1993)196:1363-1367, report the recovery of a DNA sequence which encodes a pigleukocyte antimicrobial peptide with a cathelin-like prosequence and isreported to be one of the protegrins.

Other important classes of cysteine-containing antimicrobial peptidesinclude the defensins, β-defensins and insect defensins. The defensinsare somewhat longer peptides characterized by six invariant cysteinesand three intramolecular cystine disulfide bonds. Defensins weredescribed by Lehrer, R. I. et al. Cell (1991) 64:229-230; Lehrer, R. I.et al. Ann Rev Immunol. (1993) 11:105-128. A review of mammalian-deriveddefensins by Lehrer, R. I. et al. is found in Annual Review Immunol(1993) 11:105-128; three patents have issued on the defensins: U.S. Pat.No. 4,705,777; U.S. Pat. No. 4,659,692; and U.S. Pat. No. 4,543,252.Defensins have been found in the polymorphonucleated leukocytes (PMN) ofhumans and of several other animals, as well as in rabbit pulmonaryalveolar macrophages, and in murine small intestinal epithelial (Paneth)cells and in corresponding cells in humans.

β-Defensins are found in bovine leukocytes and respiratory epithelialcells. See Selsted, M. E. et al. J. Biol Chem (1993) 288:6641-6648,Tang, Y-Q. J Biol Chem (1993) 268:6649-6653, and Diamond, G. et al.Proc. Natl Acad Sci (USA) (1991) 88:3952-3958, Diamond, G. et al. ProcNatl Acad Sci USA (1993) 90:4596-4600. Insect defensins have beenreported by Lambert, J. et al. Proc Natl Acad Sci (USA) (1989)88:262-265.

Antifungal and antibacterial peptides and proteins have also been foundin plants (Broekaert, W. F. et al. Biochemistry (1992) 31:4308-4314) asreviewed by Cornelissen, B. J. C. et al. Plant Physiol (1993)101:709-712. Expression systems for the production of such peptides havebeen used to transform plants to protect the plants against suchinfection as described, for example, by Haln, R. et al. Nature (1993)361:153-156.

Another group of antibacterial peptides which are isolated from avianleukocytes are the gallinacins. These peptides have cysteinedistribution patters which are similar to the β-defensins obtained frommammalian species. These peptides are described by Harwig, S. S. L. etal. in Techniques in Protein Chemistry V (1994) Academic Press Inc., NewYork, pages 81-88.

An interesting feature of many of the apparently unrelated naturallyoccurring antimicrobial peptides is that they are derived fromprecursors which have similar 5' regions highly homologous to a porcinecysteine proteinase inhibitor, cathelin. For example, Storici, P. et al.Biochem Biophys Res Commun (1993) 196:1363-1368, point out that theβ-defensins, as well as certain other classes of antimicrobial peptidesshare this common upstream region. These peptides include Bac5 (Gennaro,et al. Infect Immun (1989) 57:3142-3146 for which the cDNA was reportedby Zanetti, M. et al. J Biol Chem (1993) 268:522-526); indolicidin(Selsted, M. E. et al. J Biol Chem (1992) 267:4292-4295; Delsal, G. etal. Biochem Biophys Res Commun (1992) 187:467-472); an acyclicdodecapeptide from bovine sources (Romeo, D. et al. J Biol Chem (1988)263:9573-9575) for which a cDNA clone was reported by Storici, P., etal. FEBS (1992) 314:187-190); and CAP18 from rabbit leukocytes (Larrick,J. W. et al. Biochem Biophys Res Commun (1991) 179:170-175). Thisupstream sequence is also shared by the antibacterial 15 kD proteins,"p15", from rabbit polymorphonuclear leukocytes described by Levy, O. etal. J. Biol Chem (1993) 268:6058-6063. Still another member of thisfamily is the PR39 peptide described by Storici, P. et al. BiochemBiophys Res Commun (1993) 196:1058-1065. All of these are characterizedby prosequences homologous to the cysteine proteinase inhibitor,cathelin/PLCPI.

Taking advantage of this precursor homology, Pungercar, J. et al. FABS(1993) 336:284-288 (the December issue) utilized a PCR-derived probeobtained by amplification of the propeptide region of a rabbit Cap18cDNA to screen a cDNA library prepared in λ-GT11 from pig bone marrow.The retrieved cDNA clone contained a deduced amino acid sequence withthe cathelin N-terminal sequence and a putative 100-amino acid residuemature protein rich in proline. These authors further suggest that theC-terminal 3-amino acid residue may be cleaved in the mature form toyield a 97-amino acid mature protein with multiple proline residues.

The effect of proline-rich regions on the structure and function ofvarious proteins has been reviewed by Williamson, M. P. et al. Biochem J(1994) 297:249-260. Most of such proteins serve a structural or bindingpurpose since the presence of multiple proline residues imposes severeconformational constraints.

The present invention is directed to a family of proline-richantimicrobial peptides containing about 79 amino acid residues withrepeating decamer regions. The invention peptides are particularlyeffective against Gram-negative bacteria and also are capable of bindinglipopolysaccharide (LPS) thus having the ability to mitigate thesymptoms of septic shock. They are useful as preservatives andtherapeutics. This class of peptides is designated "prophenins".

DISCLOSURE OF THE INVENTION

The invention is directed to prophenins, i.e., peptides of about 79amino acid residues characterized by repeating decamer regions and amultiplicity of proline residues, as well as to fragments thereof withantimicrobial and/or LPS binding activity. These peptides can beproduced synthetically and some can be produced recombinantly or can beisolated from their native sources and purified for use as preservativesor in pharmaceutical compositions in treating or preventing infection orseptic shock in animals. Alternatively, the peptides can be formulatedinto compositions which can be applied to plants to protect them againstmicrobial infection. In still another approach, the DNA encoding thepeptides can be expressed in situ, in animals or preferably in plants,to combat infections. The peptides are also useful as standards inantimicrobial assays and in endotoxin or endotoxin inhibition assays andin binding endotoxins.

Accordingly, in one aspect, the invention is directed to a peptide inisolated and purified form of the formula: ##STR2## including theN-terminal acylated and/or C-terminal amidated or esterified formsthereof

wherein each of A₁, A₅, A₁₀, A₁₄ and A₂₀ is independently Ala, Gly orSer;

each of A₂, A₄, A₇, A₉, A₁₁, A₁₂, A₁₃, A₁₆, A₁₇, A₁₉ and A₂₂ isindependently a hydrophobic amino acid selected from the groupconsisting of Ile, Leu, Val, Phe and Met;

each of A₃ and A₈ is independently a neutral polar amino acid selectedfrom Asn and Gln or is a hydrophobic amino acid selected from the groupconsisting of Ile, Leu, Val, Phe and Met;

each of A₆, A₁₈ and A₂₁ is independently a basic amino acid selectedfrom Arg, Lys, and Har; and

wherein A₁₅ is Trp or a basic amino acid selected from Lys, Arg and Har;

wherein n is an integer of 1-6; and

the antimicrobial or LPS-binding fragments thereof.

Some of the peptides of the invention are isolatable from porcine orother mammalian leukocytes by the methods similar to those describedherein.

In still other aspects, the invention is directed to recombinantmaterials useful for the production of the peptides of the invention aswell as to cells, plants or nonhuman animals modified to containexpression systems for the production of these peptides. The inventionis also directed to pharmaceutical compositions and to compositions forapplication to plants containing the peptides of the invention as activeingredients or compositions which contain expression systems forproduction of the peptides for in situ expression of the nucleotidesequence encoding these peptides. The invention is also directed tomethods to prepare the invention peptides synthetically, to antibodiesspecific for these peptides, and to the use of the peptides aspreservatives. The invention is also directed to methods to mitigate thesymptoms of toxic shock by administering the peptides of the invention.

In other aspects, the invention is directed to the use of the compoundsof the invention as standards in antimicrobial and endotoxin binding orinhibition assays. The invention is also directed to use of theinvention compounds as preservatives for foods or other perishables.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the elution pattern of a concentrate of the ultrafiltrateof porcine leukocytes applied to an Biogel P10 column; V_(t) signifiestotal column, volume.

FIG. 2 shows the results of mass spectral determination of Prophl.

FIG. 3 shows the amino acid sequence of Prophl.

FIG. 4a shows antibacterial activity of Prophl against E. Coli;

FIG. 4b shows antibacterial activity of Prophl against Listeriamonocytogenes;

FIG. 4c shows antifungal activity of Prophl against Candida albicans.

MODES OF CARRYING OUT THE INVENTION

The prophenin peptides of the invention are generally described by theformula: ##STR3## including the N-terminal acylated and/or C-terminalamidated or esterified forms thereof, and

the antimicrobial or LPS-binding fragments thereof, where each A_(i) andn are as defined above. Those peptides of the invention which occur innature must be in purified and isolated form.

The peptides of Formula (1) or its active fragments may optionally beextended by 1-8, preferably only 1-5 and more preferably only 1-3noninterfering amino acids at either the N-terminus or C-terminus orboth.

The amino terminus of any of the peptides may be in the free amino formor may be acylated by a group of the formula RCO--, wherein R representsa hydrocarbyl group of 1-6C. The hydrocarbyl group is saturated orunsaturated and/or cyclic and is typically, for example, methyl, ethyl,i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexen-2-yl, cyclopentyl,hexen-3-yl, hexyn-4-yl, and the like.

The C-terminus of the peptides of the invention may be in the form ofthe underivatized carboxyl group, either as the free acid or anacceptable salt, such as the potassium, sodium, calcium, magnesium, orother salt of an inorganic ion or of an organic ion such as caffeine.The carboxyl terminus may also be derivatized by formation of an esterwith an alcohol of the formula ROH, or may be amidated by an amine ofthe formula NH₃, or RNH₂, or R₂ NH, wherein each R is independentlyhydrocarbyl of 1-6C as defined above. Carboxy termini of the formula--COOH and its salts or --CONH₂ are preferred. Amidated forms of thepeptides wherein the C-terminus has the formula --CONH₂ are especiallypreferred.

As the prophenin peptides of the invention contain basic amino acids,the peptides of the invention may be supplied in the form of the acidaddition salts. Typical acid addition salts include those of inorganicions such as chloride, bromide, iodide, fluoride or the like, sulfate,nitrate, or phosphate, or may be salts of organic anions such asacetate, formate, benzoate and the like. The acceptability of each ofsuch salts is dependent on the intended use, as is commonly understood.If the peptides of the invention are prepared under physiologicalconditions, the side-chain amino groups of basic amino acids will be inthe form of the relevant acid addition salts.

The peptides of the invention may contain 1-2 amino acid residues in theD, rather than in the native L form.

The amino acid notations used herein are conventional and are asfollows:

    ______________________________________                                                       One-Letter                                                                              Three-letter                                         Amino Acid     Symbol    Symbol                                               ______________________________________                                        Alanine        A         Ala                                                  Arginine       R         Arg                                                  Asparagine     N         Asn                                                  Aspartic acid  D         Asp                                                  Cysteine       C         Cys                                                  Glutamine      Q         Gln                                                  Glutamic acid  E         Glu                                                  Glycine        G         Gly                                                  Histidine      H         His                                                  Isoleucine     I         Ile                                                  Leucine        L         Leu                                                  Lysine         K         Lys                                                  Methionine     M         Met                                                  Phenylalanine  F         Phe                                                  Proline        P         Pro                                                  Serine         S         Ser                                                  Threonine      T         Thr                                                  Tryptophan     W         Trp                                                  Tyrosine       Y         Tyr                                                  Valine         V         Val                                                  ______________________________________                                    

The amino acid homoargenine, not encoded genetically, is abbreviatedHar.

In the specific peptides shown in the present application, the L-form ofany amino acid residue having an optical isomer is intended unless theD-form is expressly indicated by a dagger superscript (.sup.↑).

In all of the peptides of the invention, one or more amide linkages(--CO--NH--) may optionally be replaced with another linkage which is anisostere such as --CH₂ NH--, --CH₂ S--, --CH₂ CH₂, --CH═CH-- (cis andtrans), --COCH₂, --CH(OH)CH₂ -- and --CH₂ SO--. This replacement can bemade by methods known in the art. The following references describepreparation of peptide analogs which include these alternative-linkingmoieties: Spatola, A. F., Vega Data (Mar. 1983), Vol. 1, Issue 3,"Peptide Backbone Modifications" (general review); Spatola, A. F., in"Chemistry and Biochemistry of Amino Acids Peptides and Proteins," B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (generalreview); Morley, J. S., Trends. Pharm Sci (1980) pp. 463-468 (generalreview); Hudson, D., et al., Int J Pept Prot Res (1979) 14:177-185(--CH₂ NH--, --CH₂ CH₂ --); Spatola, A. F., et al., Life Sci (1986)38:1243 1249 (--CH₂ --S); Hann, M. M., J Chem Soc Perkin Trans I (1982)307-314 (--CH--CH--, cis and trans); Almquist, R. G., et al., J Med Chem(1980) 23:1392-1398 (--COCH₂ --); Jennings-White, C., et al.,Tetrahedron Lett (1982) 23:2533 (--COCH₂ --); Szelke, M., et al.,European Application EP 45665 (1982) CA:97:39405 (1982) (--CH(OH)CH₂--); Holladay, M. W., et al., Tetrahedron Lett (1983) 24:4401-4404(--C(OH) CH₂ --); and Hruby, V. J., Life Sci (1982) 31:189-199 (--CH₂--S--).

In an alternative embodiment, the peptides of the invention are definedas described by Formula (1) but wherein the specific peptide in eachcase is determined by the isolatability of the peptide from mammalianleukocytes, preferably porcine leukocytes, by the invention method. Theinvention method comprises the steps of providing an ultrafiltrate of alysate of porcine leukocytes and isolating antimicrobial peptides ofabout 79 amino acids. These peptides can further be defined by theability of DNA encoding them to hybridize under stringent conditions tothe DNA from porcine leukocytes encoding the peptide exemplified asProphl herein.

Preferred Embodiments

In general, peptides of Formula (1) wherein each of A₂, A₄, A₇, A₉, A₁₁,A₁₃, A₁₆, A₁₇, A₁₉ and A₂₂ is independently selected from the groupconsisting of Val, Ile and Phe are preferred. Also preferred are thosewherein each of A₅, A₁₀, A₁₄ and A₂₀ is Gly and A₁ is Ala or whereineach of A₆, A₁₈ and A₂₁ is Arg or wherein A₁₅ is Trp or wherein n is 4.Especially preferred are those embodiments wherein A₁₂ is Val or Ile andA₂, A₇, A₉, A₁₁, A₁₃, A₁₆, A₁₇, and A₁₉ are Phe or Val and especiallywherein A₂, A₇, A₁₁ and A₁₆ are Phe.

Particularly preferred compounds of the invention are selected from thegroup consisting of ##STR4## wherein the C-terminal carboxy is --COOH orits salts or is --CONH₂, especially Prophl.

Active fragments of the invention include those of the formula selectedfrom the group consisting of: ##STR5## the composites thereof. Thus,each of the above fragments may be used per se or may be ligated to anidentical fragment or to a different fragment up to a total of 79 aminoacids.

Preparation of the Invention Compounds

Standard methods of synthesis of peptides are known. Most commonly usedcurrently are solid phase synthesis techniques; indeed, automatedequipment for systematically constructing peptide chains can bepurchased. Solution phase synthesis can also be used but is considerablyless convenient. When synthesized using these standard techniques, aminoacids not encoded by gene, and/or D-enantiomers can be employed in thesynthesis. Thus, one very practical way to obtain the compounds of theinvention is to employ these standard chemical synthesis techniques.

In addition to providing the peptide backbone, the N- and/or C-terminuscan be derivatized, again using conventional chemical techniques. Thecompounds of the invention may optionally contain an acyl group,preferably an acetyl group at the amino terminus. Methods foracetylating or, more generally, acylating, the free amino group at theN-terminus are generally known in the art; in addition, the N-terminalamino acid may be supplied in the synthesis in acylated form.

At the carboxy terminus, the carboxyl group may of course, be present inthe form of a salt; in the case of pharmaceutical compositions this willbe a pharmaceutically acceptable salt. Suitable salts include thoseformed with inorganic ions such as NH₄ ⁺, Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, and thelike as well as salts formed with organic cations such as those ofcaffeine and other highly substituted amines. The carboxy terminus mayalso be esterified using alcohols of the formula ROH wherein R ishydrocarbyl (1-6C) as defined above. Similarly, the carboxy terminus maybe amidated so as to have the formula --CONH₂, --CONHR, or --CONR₂,wherein each R is independently hydrocarbyl (1-6C) as herein defined.Techniques for esterification and amidation as well as neutralizing inthe presence of base to form salts are all standard organic chemicaltechniques.

If the peptide backbone is comprised entirely of gene-encoded aminoacids, or if some portion of it is so composed, the peptide or therelevant portion may also be synthesized using recombinant DNAtechniques. A nucleic acid molecule encoding the peptides of theinvention may itself be synthesized using commercially availableequipment; codon choice can be integrated into the synthesis dependingon the nature of the host. Alternatively, although less convenient, anencoding DNA can be obtained, at least initially, by screening a cDNAlibrary prepared from porcine leukocytes using probes or PCR primersbased on the amino acid sequences of the prophenins described herein.This results in recovery of the naturally occurring sequence encodingthe prophenins of the invention. Obtention of this native sequence issignificant for purposes other than the synthesis of the prophenins perse; the availability of the naturally occurring sequences provides auseful probe or permits the design of an appropriate amplificationprimer to obtain corresponding active DNA encoding prophenins of otherspecies. Thus, cDNA libraries, for example, of leukocytes derived fromother animals can be amplified using these primers or screened using thenative DNA, preferably under conditions of high stringency. Highstringency is as defined by Maniatis, et al. Molecular Cloning: aLaboratory Manual 2nd Ed, Cold Spring Harbor Laboratory Press (1989),the relevant portions of which are incorporated herein by reference.These procedures also permit recovery of allelic variants of thesepeptides from the same species.

Alternatively, the prophenins can be prepared by isolation fromleukocytes of an animal species using techniques similar to thosedisclosed herein for the isolation of porcine prophenins. In general,these techniques involve preparing a lysate of a leukocyte preparation,ultrafiltering the supernatant of the clarified lysate and recoveringthe ultrafiltrate. The ultrafiltrate is then subjected tochromatographic separation. The location of fragments havingantimicrobial activity corresponding to prophenins can be assessed usingcriteria of molecular weight and assaying the fractions for the desiredactivities as described herein.

Isolated and recombinantly produced forms of the prophenins may requiresubsequent derivatization to modify the N- and/or C-terminus. Dependingon the host organism used for recombinant production and the animalsource from which the protein is isolated, some or all of theseconversions may already have been effected.

For recombinant production, the nucleotide sequence encoding theprophenins of the invention is included in an expression system whichplaces these coding sequences under control of a suitable promoter andother control sequences compatible with an intended host cell. Types ofhost cells available span almost the entire range of the plant andanimal kingdoms. Thus, the prophenins of the invention could be producedin bacteria or yeast (to the extent that they can be produced in anontoxic or refractile form or utilize resistant strains) as well as inanimal cells, insect cells and plant cells. Indeed, modified plant cellscan be used to regenerate plants containing the relevant expressionsystems so that the resulting transgenic plant is capable of selfprotection vis-a-vis these infective agents. In principle, transgenicnonhuman animals containing these expression systems could also beobtained. In this embodiment, generally, viral control sequences areuseful.

The prophenins of the invention can be produced recombinantly in a formthat will result in their secretion from the host cell by fusing to thenucleotide sequence encoding the prophenin, a nucleotide sequenceencoding a suitable signal peptide. The prophenin may also be producedintracellularly. Prophenins may also be produced as fusion proteins withadditional amino acid sequence which may or may not need to besubsequently removed prior to the use of these compounds asantimicrobials.

Thus, the prophenins of the invention can be produced in a variety ofmodalities including chemical synthesis, recombinant production,isolation from natural sources, or some combination of these techniques.

In particular, the active fragments of the prophenins can be preparedand used as fragments per se, or can be ligated together as composites.For example, fragment (1a) may be ligated to one or two additional (1a)fragments, or to (1b) or to (1c) or to (1e) or to multiples thereof.

Those members of the prophenin class which occur naturally are suppliedin purified and isolated form. By "purified and isolated" is meant freefrom the environment in which the peptide normally occurs (in the caseof such naturally occurring peptides) and in a form where it can be usedpractically. Thus, "purified and isolated" form means that the peptideis substantially pure, i.e., more than 90% pure, preferably more than95% pure and more preferably more than 99% pure or is in a completelydifferent context such as that of a pharmaceutical preparation.

Antibodies

Antibodies to the prophenins of the invention may also be produced usingstandard immunological techniques for production of polyclonal antiseraand, if desired, immortalizing the antibody-producing cells of theimmunized host for sources of monoclonal antibody production. Techniquesfor producing antibodies to any substance of interest are well known. Itmay be necessary to enhance the immunogenicity of the substance,particularly as here, where the material is only a short peptide, bycoupling the hapten to a carrier. Suitable carriers for this purposeinclude substances which do not themselves produce an immune response inthe mammal to be administered the hapten-carrier conjugate. Commoncarriers used include keyhole limpet hemocyanin (KLH), diphtheriatoxoid, serum albumin, and the viral coat protein of rotavirus, VP6.Coupling of the hapten to the carrier is effected by standard techniquessuch as contacting the carrier with the peptide in the presence of adehydrating agent such as dicyclohexylcarbodiimide or through the use oflinkers such as those available through Pierce Chemical Company,Chicago, Ill.

The prophenins of the invention in immunogenic form are then injectedinto a suitable mammalian host and antibody titers in the serum aremonitored. Polyclonal antisera may be harvested when titers aresufficiently high. Alternatively, antibody-producing cells of the hostsuch as spleen cells or peripheral blood lymphocytes may be harvestedand immortalized. The immortalized cells are then cloned as individualcolonies and screened for the production of the desired monoclonalantibodies.

The antibodies of the invention are, of course, useful in immunoassaysfor determining the amount or presence of the prophenins. Such assaysare essential to quality controlled production of compositionscontaining the prophenins of the invention. In addition, the antibodiescan be used to assess the efficacy of recombinant production of theprophenins, as well as screening expression libraries for the presenceof prophenin encoding genes.

Compositions Containing the Prophenins and Methods of Use

The prophenins of the invention are effective in inactivating a widerange of microbial targets, including Gram-positive and Gram-negativebacteria and yeast but are particularly highly specific forGram-negative bacteria. Accordingly, they can be used in disinfectantcompositions and as preservatives for materials such as foodstuffs,cosmetics, medicaments, or other materials containing nutrients fororganisms. For use in such contexts, the prophenins (or fragments orcomposites) are supplied either as a single prophenin (or fragment orcomposite), in admixture with several other prophenins (or fragments orcomposites), or in admixture with additional antimicrobial agents. Ingeneral, as these are preservatives in this context, they are usuallypresent in relatively low amounts, of less than 5%, by weight of thetotal composition, more preferably less than 1%, still more preferablyless than 0.1%.

The peptides of the invention are also useful as positive controls inantimicrobial assays, especially for assays against Gram-negativebacteria.

For use as antimicrobials in the treatment of animal subjects, theprophenins and/or fragments and/or composites can be formulated aspharmaceutical or veterinary compositions. Depending on the subject tobe treated, the mode of administration, and the type of treatmentdesired--e.g., prevention, prophylaxis, therapy; the prophenins andrelated peptides are formulated in ways consonant with these parameters.A summary of such techniques is found in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co., Easton, Pa.

The prophenins and/or fragments and/or composites can be administeredsingly or as mixtures of several prophenins, and/or fragments and/orcomposites, or in combination with other pharmaceutically activecomponents. The formulations may be prepared in a manner suitable forsystemic administration or topical or local administration. Systemicformulations include those designed for injection (e.g., intramuscular,intravenous or subcutaneous injection) or may be prepared fortransdermal, transmucosal, or oral administration. The formulation willgenerally include a diluent as well as, in some cases, adjuvants,buffers, preservatives and the like. The peptides of the invention canbe administered also in liposomal compositions or as microemulsions.

If administration is to be oral, the peptides of the invention must beprotected from degradation in the stomach using a suitable entericcoating. This may be avoided to some extent by utilizing amino acids inthe D-configuration, thus providing resistance to proteases. However,the peptide is still susceptible to hydrolysis due to the acidicconditions of the stomach; thus, some degree of enteric coating maystill be required.

The peptides of the invention may also be applied to plants or to theirenvironment to prevent microbial-induced diseases in these plants.Suitable compositions for this use will typically contain a diluent aswell as a spreading agent or other ancillary agreements beneficial tothe plant or to the environment.

Thus, the peptides of the invention may be used in any context whereinan antimicrobial action is required. This use may be an entirely invitro use, or the peptides may be administered to organisms.

In addition, the antimicrobial activity may be generated in situ byadministering an expression system suitable for the production of thepeptides of the invention. Such expression systems can be supplied toplant and animal subjects using known techniques. For example, inanimals, pox-based expression vectors can be used to generate thepeptides in situ. Similarly, plant cells can be transformed withexpression vectors and then regenerated into whole plants which arecapable of their own production of the peptides.

As shown hereinbelow, the prophenins and their fragments are capable ofinactivating endotoxins derived from Gram-negative bacteria--i.e., thelipopolysaccharides (LPS)--in standard assays. Accordingly, the peptidesof the invention may be used under any circumstances where inactivationof LPS is desired. One such situation is as a positive control in testsfor LPS binding, analogous to the manner in which polymyxin-B is used inExample 3 hereinbelow. Another such situation is in the treatment oramelioration of Gram-negative sepsis.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Purification of Prophenin

Fresh porcine blood was collected at an abattoir into 15 liter vesselsthat contained an anticoagulant, 5% EDTA in normal saline, pH 7.4 (33ml/L blood). After the blood cells had sedimented spontaneously for 90min at room temperature, the leukocyte-rich supernatant was removed andcentrifuged at 200×g for 5-7 min. The sediments were pooled andsuspended in 0.84% ammonium chloride to lyse the erythrocytes. Theresulting leukocytes (70-75% PMN, 5-10% eosinophils, 15-25% lymphocytesand monocytes) were washed in normal saline, resuspended in ice cold 10%acetic acid at 10⁸ cells/ml, homogenized and stirred overnight at 4° C.This preparation was centrifuged at 25,000×g for 3 h at 4° C. and itssupernatant was lyophilized and weighed.

Lyophilized extract (950 mg dry weight) contained 520 mg of protein byBCA analysis. It was stirred overnight at 4° C. in 100 ml of 10% aceticacid, and then centrifuged at 25,000×g for 2 h. the supernatant wasremoved and passed by pressure through a 50 ml stirred ultrafiltrationcell (Amicon, Danvers, Mass.) that contained YM-5 filter. Theultrafiltrate (24.5 mg of protein by BCA) was concentrated to 3 ml byvacuum centrifugation (Speed Vac Concentrator, Savant Instruments,Hicksville, N.Y.), applied to a 2.5×117 cm column of BioGel P-10(BioRad, Hercules, Calif.), eluted at 4° C. with 5% acetic acid, andcollected in 6.6 ml fractions.

Aliquots (66 μl ) of each fraction were dried by vacuum centrifugation,and resuspended in approximately 6.6 μl of 0.01% acetic acid. Five μlsamples of this concentrate were tested for antimicrobial activityagainst E. coli ML-35, L. monocytogenes, Strain EGD and C. albicans,strain 820 by radial diffusion and gel overlay techniques (Lehrer, R. I.et al. J Immunol Meth (1991) 137:167-173). Active fractions were furtherexamined by acid-urea PAGE and SDS-PAGE. Prophenins emerged from the P10column in fractions 38-43 (39.6 ml total volume), which contained atotal of 18 mg protein BCA analysis. The elution curve is shown in FIG.1.

The prophenins eluted late (≈48% acetonitrile from a C-18 RP-HPLCcolumn, well after the majority of other leukocyte-derived peptides hademerged. SDS-PAGE indicated a size of 6-7 kDa and AU-PAGE showed thepresence of at least two distinct molecular species whose overall aminoacid composition was approximately 44% proline, 14% phenylalanine and 8%arginine. After final purification, approximately 400 μg of prophenin-1and 1 mg of "prophenin-2" were recovered. Prophenin-1, the more cationicform, was purified by preparative CAU-PAGE, followed by RP-HPLC, andappeared homogeneous on AU-PAGE and SDS-PAGE and was virtuallyhomogeneous on electrospray-mass spectroscopy. The mass of Prophenin:was 8,683 by ESI-MS, consistent with its calculated mass (8685) from thesequence shown herein. The minor peak (mass 8613) represented des Ala₁-prophenin-1. These data are shown in FIG. 2.

Although Prophenin-2 appeared homogeneous by AU-PAGE, SDS-PAGE andRP-HPLC, ESI-MS analysis revealed the presence of two species, withmasses of 8,807 and 8,794. "Prophenin-2" is thus a mixture of twoclosely related prophenins that differ in primary sequence from eachother and from prophenin-1. The amino acid analysis of the "Prophenin-2"mixture showed that none of Cys, Ser, His, Tyr, Met, Leu and Lys werepresent; Trp was not determined.

    ______________________________________                                        Amino acid analysis of "Prophenin-2"                                                                               Comments                                        pmols      mols %   Residues  relative                                 Residue                                                                              detected   detected per 78 res.                                                                             to PF-1                                  ______________________________________                                        Asx    1.365      6.78     5.3                                                Glx    0.356      1.77     1.4       more                                     Gly    2.312      11.48    9.0                                                Arg    2.104      10.45    8.2       more                                     Ala    0.265      1.32     1.0                                                Pro    9.784      48.6     37.9      less                                     Val    0.310      1.54     1.2                                                Ile    0.200      0.99     0.8                                                Phe    3.433      17.1     13.3      more                                     ______________________________________                                    

Amino acid sequences were determined by gas-phase Edman degradation witha Porton Model 2090 instrument, using purified a 1 nmol sample ofprophenin-1. The initial yield was 57.6% (576 pmols) and the repetitiveyield was 95.98%. The initial sequencing of native prophenin-1 providedunambiguous identification of all but 3 of its initial 65 residues.

To confirm these data and ascertain the remaining residues, a trypticdigest was prepared by incubating 17 μg of prophenin-1 in 30 μl of 0.1Mammonium bicarbonate, pH 8.15, with 0.3 μg of TPCK-treated trypsin(Worthington) for up to 3 h at 37° C. The digestion was stopped byadding glacial acetic acid (final conc. 25%), an aliquot was removed foron-line capillary-LC-ESI-MS and the remainder was purified by RP-HPLC ona Vydac C-18 column with a gradient of acetonitrile in 0.1%trifluoroacetic acid. Electrospray ionization (ESI)-mass spectrometricanalyses were performed by Kristine Swiderek and Terry D. Lee at theDivision of Immunology, Beckman Research Institute of the City of Hope,Duarte, Calif. The sample (approximately 30 pmols) was analyzed asdescribed by Harwig, S. et al. in Techniques in Protein Chem V (1994) J.Crabb, ed. Academic Press, N.Y. pp. 81-88.

The trypsin digest eluted as three broad peaks in Fractions 1, 2 and 3in the order of their elution from the column. Fraction 1 contained amajor component with a mass of 1148, and a minor component with a massof 1077. It corresponded to an undecamer composed of residues 1-11, witha minor amount of the decamer composed of residues 2-11. Fraction 2 hada mass of 1125 and was about 3× more abundant than Fraction 1. Itcorresponded to the repeating FPPPNFPGPR decamer found in residues12-41. Fraction 3 had a mass of 3986. By microsequencing through toresidue 76 (proline), we determined that it corresponded to residues42-77 of prophenin-1. The presence of arginine-77 was confirmed by aminoacid analysis and the mass measurement. The amino acid sequence ofprophenin-1 is shown in FIG. 3.

EXAMPLE 2 Antimicrobial Activity

The radial diffusion assay in agarose gels as described above was alsoused to test the activity of the purified prophenins. Samples of 5 μLeach were added to 3 mm wells in thin gels composed of 10 mM sodiumphosphate, 30 mg/ml trypticase soy broth powder in 1% agarose andcontaining approximately 4×10⁵ CFU/ml of mid-log phase organisms. Afterallowing 3 hours for the samples to diffuse into the underlayer gel, anutrient-rich overlay gel was poured and the plates were incubated for24 hours at 37° C. to allow surviving organisms to form microcolonies.The diameters of the clear zones were measured and expressed in "units"(10 units=1 mm) as described previously.

FIGS. 4a, 4b and 4c show the results against three test organisms inunits described as above. Rabbit defensin (NP-1) and human defensin(HNP-1) and protegrin-3 (PG3) were used as controls.

FIG. 4a shows that prophenin-1 (closed circles) was comparable ininhibitory activity to PG3 and NP1 against E. coli ML-35. All of thesewere more inhibitory than HNP-1.

FIG. 4b shows that prophenin-1 was substantially less inhibitory againstL. monocytogenes than were the other three antibiotic peptides. However,some activity was shown at concentrations above 100 μg/ml.

FIG. 4c shows that prophenin-1, like HNP-1, is substantiallynoninhibitory at reasonable concentrations against C. albicans. Theother two antibiotics tested, PG3 and NP1 were successful antifungals.

Quantitative results are shown in Table 2 which gives the minimalmicrobicidal concentration for each of the peptides tested in μg/ml.

                  TABLE 2                                                         ______________________________________                                        Target        PF-1    HNP-2     NP-1 PG-3                                     ______________________________________                                        E. coli       15.2    80.0      11.5 8.6                                      L. monocytogenes                                                                            122.2   12.0      3.4  9.0                                      C. albicans   >150    >150      3.7  6.9                                      ______________________________________                                    

These results show that, unlike the other three peptides tested,prophenin-1 is highly specific for Gram-negative bacteria.

EXAMPLE 3 Ability to Bind Endotoxin

The prophenins of the invention were tested for their ability to bindthe lipid polysaccharide (LPS) of the Gram-negative bacterium E. colistrain 0.55B5. The assay was the Limulus amebocyte lysate (LAL) test forendotoxins conducted in the presence and absence of the test compounds.The test was conducted using the procedure described in Sigma TechnicalBulletin No. 210 as revised in Dec. 1992 and published by Sigma ChemicalCompany, St. Louis, Mo.

The LAL test is based on the ability of LPS to effect gelation in thecommercial reagent E-TOXATE™ which is prepared from the lysate ofcirculating amebocytes of the Horseshoe Crab Limulus polyphemus. Asdescribed in the technical bulletin, when exposed to minute quantitiesof LPS, the lysate increases in opacity as well as viscosity and may gelor clot depending on the concentration of endotoxin. The technicalbulletin goes on to speculate that the mechanism appears analogous tothe clotting of mammalian blood and involves the steps of activation ofa trypsin-like preclotting enzymes by the LPS in the presence of calciumion, followed by enzymic modifications of a "coagulogen" by proteolysisto produce a clottable protein. These steps are believed tied to thebiologically active or "pyrogenic" portion of the molecule. It has beenshown previously that detoxified LPS (or endotoxin) gives a negative LALtest.

Polymyxin B is a cyclic 1.2 kD acylated nonapeptide obtained fromBacillus polymyxa. Polymyxin B was used as a positive control since itis known to inhibit LPS-induced Limulus hemocyte lysate clotting.

In the assay, the amounts of prophenin-1 and polymyxin B (78% freepolymyxin, Sigma) were preincubated in a final volume of 0.2 ml and thetest mixtures contained LPS at a final concentration of 0.05 endotoxinunit/ml and E-TOXATE™ at the same concentration. The test compounds wereincubated together with the LPS for 15 minutes before the E-TOXATE™ wasadded to a final volume (after E-TOXATE™ addition) of 0.2 ml. The tubeswere then incubated for 60 minutes at 37° C. and examined for theformation of a gel.

In Table 3, ++ signifies a solid gel (no inhibition), + indicatesincreased viscosity without solid gel formation (partial inhibition) and0 indicates that gelation was absent (complete inhibition).

                  TABLE 3                                                         ______________________________________                                        Peptide (μg/ml)                                                                      10.0     5.0   2.5   1.25 0.63 0.31 0.16                            ______________________________________                                        Polymyxin B                                                                             0        0     +     ++   ++   ++   ++                              Prophenin-1                                                                             not done 0     0     0    0    ++   ++                              ______________________________________                                    

Complete inhibition was obtained with 5 μg/ml (≈4.2 μM) polymyxin Bsulfate and 0.625 μg/ml (≈72 nM) prophenin-1; thus, on a molar basis,Prophenin-1 is approximately 60-fold more potent than polymyxin Bsulfate (4.2/0.072) in binding LPS.

The ability of fragments of prophenin-1 to bind LPS was also assayed.Prophenin-1 was digested with TLCK treated trypsin as described above,thus cleaving the protein C-terminal to the initial 4 arginines. Thefragments were purified by RP-HPLC, and their identities were verifiedby ESI-MS and amino acid composition analysis. The three fragments werethose obtained as described in Example 1 and are as follows:

Fraction 1: Positions 1-11:

AFPPPNVPGPR

Fraction 2: Positions 12-21, 22-31, or 32-41:

FPPPNFPGPR

Fraction 3: Positions 42-77:

FPPPNFPGPPFPPPIFPGPWFPPPPPFRPPPFGPPR.

The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        μg/ml                                                                              10.0   5.0     2.5 1.25   .625 .31    .16                             ______________________________________                                        Fraction 1                                                                            0      0       +   ++     ++   ++     ++                              Fraction 2                                                                            0      0       +   +      ++   ++     ++                              Fraction 3                                                                            0      +       +   +      ++   ++     ++                              ______________________________________                                    

Taking into account the molecular masses of the respective Fractions,the data indicate that Fractions 1, 2 and 3 were each effective asbetween 2-μM, (approximately equivalent polymyxin B on a molar basis) ininhibiting LPS, whereas intact prophenin-1 molecule shows an effectiveconcentration of 35-70 nM. Thus, the subunits of prophenin-1 appear tobe useful in binding LPS although the holoprotein is more effective, andindeed more effective than the additive value of the subunits.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaPheProProProAsnValProGlyProArgPheProProProAsn                              151015                                                                        PheProGlyProArgPheProProProAsnPheProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProArgPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProTrpPheProPro                              505560                                                                        ProProProPheArgProProProPheGlyProProArgPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaPheProProProAsnValProGlyProArgPheProProProAsn                              151015                                                                        PheProGlyProArgPheProProProAsnIleProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProArgPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProTrpPheProPro                              505560                                                                        ProProProPheArgProProProPheGlyProProArgPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaPheProProProAsnValProGlyProArgPheProProProAsn                              151015                                                                        PheProGlyProArgPheProProProAsnPheProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProArgPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProArgPheProPro                              505560                                                                        ProProProPheArgProProProPheGlyProProArgPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 41                                                              (D) OTHER INFORMATION: /note="This position is Har."                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaPheProProProAsnValProGlyProArgPheProProProAsn                              151015                                                                        PheProGlyProArgPheProProProAsnPheProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProAsnPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProTrpPheProPro                              505560                                                                        ProProProPheArgProProProPheGlyProProLysPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaPheProProProAsnValProGlyProArgPheProProProGln                              151015                                                                        PheProGlyProArgPheProProProAsnPheProGlyProArgPhe                              202530                                                                        ProProProGlnPheProGlyProArgPheProProProPheProGly                              354045                                                                        ProProPheProProProValPheProGlyProTrpPheProProPro                              505560                                                                        ProProPheArgProProProPheGlyProProArgPhePro                                    657075                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 69                                                              (D) OTHER INFORMATION: /note="This position is Har."                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaPheProProProAsnValProGlyProLysPheProProProAsn                              151015                                                                        IleProGlyProArgPheProProProAsnIleProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProArgPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProTrpPheProPro                              505560                                                                        ProProProPheAsnProProProPheGlyProProArgPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaPheProProProAsnValProGlyProArgValProProProAsn                              151015                                                                        PheProGlyProArgPheProProProAsnPheProGlyProPhePro                              202530                                                                        ProProAsnPheProGlyProArgIleProProProAsnPheProGly                              354045                                                                        ProProPheProProProIlePheProGlyProTrpPheProProPro                              505560                                                                        ProProPheArgProProProPheGlyProProArgPhePro                                    657075                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaPheProProProAsnPheProGlyProArgPheProProProAsn                              151015                                                                        PheProGlyProArgValProProProAsnPheProGlyProArgPhe                              202530                                                                        ProProProAsnPheProGlyProArgPheProProProAsnPhePro                              354045                                                                        GlyProProPheProProProIlePheProGlyProTrpPheProPro                              505560                                                                        ProProProPheArgProProProPheGlyProProArgPhePro                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       PheProProProAsnPheProGlyProProPheProProProIlePhe                              151015                                                                        ProGlyProTrpPheProProProProProPheArgProProProPhe                              202530                                                                        GlyProProArg                                                                  35                                                                            __________________________________________________________________________

We claim:
 1. A method to inhibit the growth of a microbe which methodcomprises contacting a composition which supports the growth of saidmicrobe with a peptide comprising the amino acid sequenceAFPPPNVPGPRFPPPNFPGPR FPPPNFPGPR FPPPNFPGPR FPPPNFPGPP FPPPIFPGPW FPPPPPFRPPPFGPPRFP SEQ ID NO: 1or antimicrobial fragments thereof comprising atleast 10 contiguous amino acid residues in an amount effective toprevent said growth.
 2. The method of claim 1 wherein said microbe iscapable of growing in a foodstuff.
 3. The method of claim 1 wherein saidmicrobe is Gram-negative.
 4. A method to inactivate the endotoxin ofGram-negative bacteria, which method comprises contacting said endotoxinwith a peptide comprising the amino acid sequenceAFPPPNVPGPR FPPPNFPGPRFPPPNFPGPR FPPPNFPGPR FPPPNFPGPP FPPPPPFRPP PFGPPRFP SEQ ID NO: 1orLPS-binding fragments thereof comprising at least 10 contiguous aminoacid residues in an amount effective to inactivate said endotoxin. 5.The method of claim 4 wherein said contacting is in a subject harboringGram-negative bacteria.